Summary The goal of this Project is to understand the structural basis and mechanism of HIV-1 genome recognition and cytoplasmic trafficking, and the role of the genome in nucleating virus assembly. The Project brings together several highly collaborative groups with a broad range of expertise, and is tightly integrated with the NMR, cryoEM, X-ray crystallography, and Computational Cores. Pornillos has contributed significantly to our understanding of how the highly conserved SP1 domain of Gag, which bridges the capsid and nucleocapsid (NC) domains, and forms a hexameric coiled-coil that helps drive particle formation. Summers and Telesnitsky have recently determined the structure of the RNA element that is responsible for RNA packaging (?CES) and shown how transcriptional start site heterogeneity contributes to genome packaging. These investigators will combine efforts to determine how Gag recognizes and assembles on the RNA packaging signal. Exciting preliminary studies reveal that hexameric Gag constructs that minimally include the SP1 and NC domains bind with sub-nM affinity to the dimeric packaging signal [?CES]2 with 6:2 protein:RNA stoichiometry, and that binding is selective for the dimeric (rather than monomeric) RNA. Thus, a single Gag hexamer binds with high affinity to the dimeric RNA that contains two core encapsidation elements, an unexpected finding! This stoichiometry is consistent with confocal imaging studies in living, transfected cells by Simon, which show that the genome is anchored to the plasma membrane by a small number of (fewer than a dozen) Gag proteins. With Ganser-Pornillos of the CHEETAH Center, cryoEM studies will be conducted with complexes formed between [?CES]2 and Gag constructs containing SP1-NC. Mechanistic hypotheses will be tested by packaging and assembly experiments conducted by Telesnitsky. In addition, Simon has developed a new imaging system with which he has observed RNA dimers in the nucleus and found that most genomes arrive at the plasma membrane as dimers. Both of these preliminary findings challenge current dogma and point to additional studies. The stage is now set to (1) determine the primary site(s) of genome dimerization and Gag binding in infected cells, (2) monitor the pathway(s) that the genome follows from the time it is synthesized in the nucleus until it buds from the plasma membrane, (3) understand the molecular mechanisms that drive Gag:RNA assembly, and (4) determine the 3D structure of the ribonucleoprotein complex that anchors the dimeric genome to the plasma membrane and nucleates virus assembly.